When a plurality of enzymes attack their substrates at different sites and create new sites for each other, whereby the resulting activity is higher than the sum of the individual activities, they are said to act synergistically. Enhancing enzyme activity in order to improve industrial processes is one of the most important biotechnological and industrial challenges of recent years. In this context, one group of enzymes that has received much attention is the carbohydrate active enzymes, which is a large group of enzymes that catalyze the breakdown, biosynthesis or modification of carbohydrates and glycoconjugates. Members of this group play an important role in the degradation of cellulosic biomass to soluble sugars that can be converted by microorganisms into liquid fuels, and are therefore of great interest in the fields of bio-energy and bio-fuel production.
The broad group of carbohydrate active enzymes is divided into enzyme classes and further into enzyme families according to a standard classification system (Cantarel et al. 2009 Nucleic Acids Res 37:D233-238). According to this classification system, four enzyme classes are defined, namely glycoside hydrolases, glycosyl transferases, polysaccharide lyases and carbohydrate esterases. Each class includes various enzymatic activities and substrate specificities and is further divided into families numbered in ascending order based on sequence similarities. The different families within each class may display a very broad diversity. For example, one family may contain members from bacteria, fungi, plants and animals, with several different activities and substrate specificities. In addition, a certain activity (for example, a xylanase) may be found in several different families. An informative and updated classification of carbohydrate active enzymes is available on the Carbohydrate-Active Enzymes (CAZy) server.
Typically, carbohydrate active enzymes are characterized by a multi-modular organization, where the catalytic module is associated with one or more ancillary, helper, modules which modulate the enzyme activity. Each module or domain comprises a consecutive portion of the polypeptide chain and forms an independently folding, structurally and functionally distinct unit. One of the main ancillary modules is the carbohydrate-binding module.
The complex structure of cellulosic materials (which may include, for example, cellulose, hemicellulose, lignin) requires cooperation of many types of carbohydrate active enzymes for degradation. Enhancing synergy between carbohydrate active enzymes could lead to improved degradation, and thus has a great impact in the field of energy production from cellulosic biomass.
Synergism has been demonstrated previously between different types of carbohydrate active enzyme, in particular between glycoside hydrolases such as cellulases and xylanases. For example, synergism has been demonstrated between cellulases from different microbial systems (Irwin et al. 1993 Biotechnol. Bioeng. 42:1002-1013), between cellulosomal and non-cellulosomal enzymes (Murashima et al. 2003 J. Bacteriol. 185(5):1518-24, Koukiekolo et al. 2005 Appl Environ Microbiol. 71(7):3504-11), between different types of enzymes from different families and between enzymes that have different substrate-degradation mechanisms (i.e. exoglucanase and endoglucanase).
As another example, synergism has been demonstrated among glycoside hydrolases from the bacterium Termobifida fusca, for example, between xylan-degrading enzymes (Bachmann et al. 1991 Appl. Environ. Microbiol. 57:2121-2130, Tuncer et al. 2003 J Appl Microbiol. 94(6):1030-5) and between cellulose-degrading enzymes (Wilson et al. 2004 Chem. Rec. 4:72-82). Attempts in enhancing T. fusca enzyme synergism have been undertaken while integrating its enzymes into designer cellulosomes (Caspi et al. 2006 Biocat. Biotransform. 24:3-12).
The cellulosome system is a multi-enzyme complex characterized by a strong bi-modular protein-protein interaction between “cohesin” and “dockerin” modules that integrates the various enzymes into the complex. “Scaffoldin” subunits (non-enzymatic protein components) contain the cohesin modules that incorporate the enzymes into the complex via their resident dockerins. The primary scaffoldin subunit also includes a carbohydrate (cellulose)-binding module (CBM) through which the complex recognizes and binds to the cellulosic substrate. Previous research has suggested that the multi-enzyme cellulosome complex from the bacterium Clostridium thermocellum is far more efficient than free cellulase systems that were tested in degrading polysaccharides.
The designer cellulosome concept is based on the very high affinity and specific interaction between cohesin and dockerin modules from the same microorganism species. Since the various scaffoldin-borne cohesins of a given species essentially show the same specificity of binding for the enzyme-borne dockerins, designer cellulosomes are constructed from recombinant chimeric scaffoldins containing divergent cohesins from different species to which matching dockerin-containing enzyme hybrids are prepared. In effect, in designer cellulosomes, enzymes are complexed together on a scaffoldin subunit via the very strong and specific cohesin-dockerin interaction. In such designer cellulosome complexes, enzyme proximity, combined with substrate binding via a carbohydrate-binding module contained in the scaffoldin, resulted in enhanced enzymatic activities in several cases, for example, as described in Fierobe et al. 2005 J. Biol. Chem. 280:16325-16334.
Apart from the designer cellulosome approach, another attempt to increase enzyme synergism has been reported in the form of multifunctional enzyme conjugates. An increase in degradation of natural substrates was observed upon fusing two or three complementary xylan-degrading activities (xylanase, arabinofuranosidase and xylosidase) into the same polypeptide chain. This approach may be cost-reducing, however, strategies involving multifunctional enzyme are limited to small numbers of enzymes and restricted to sub-optimal equimolar ratios of enzymes.
International Patent Application Publication No. WO 1997/014789 discloses an enzymatic array, which composition comprises one or more enzymes non-covalently bound to a peptide backbone, wherein at least one of the enzymes is heterologous to the peptide backbone and the peptide backbone is capable of having bound thereto a plurality of enzymes. The array is useful, for example, in recovery systems, targeted multi-enzyme delivery systems, soluble substrate modification, quantification type assays, and other applications in the food industry, feed, textiles, bioconversion, pulp and paper production, plant protection and pest control, wood preservatives, topical lotions and biomass conversions.
International Patent Application Publication No. WO 2010/057064 discloses designer cellulosomes for efficient hydrolysis of cellulosic material and more particularly for the generation of ethanol.
International Patent Application Publication No. WO 2010/096562 discloses the engineering and expression of heterologous cellulosomes in microorganisms in order to facilitate the conversion of biomass to useful products. In some embodiments, the invention relates to the expression of scaffoldin proteins which form the nucleus of a cellulosome. Cellulases or other biomass-degrading enzymes can be non-covalently linked to the scaffoldin protein by virtue of a dockerin domain-cohesin domain interaction.
U.S. Patent Application Publication Nos. 2009/0155238 and 2011/0016545 disclose enzymes having xylanase, mannanase and/or glucanase activity, e.g., catalyzing hydrolysis of internal β-1,4-xylosidic linkages or endo-β-1,4-glucanase linkages; and/or degrading a linear polysaccharide β-1,4-xylan into xylose. Methods and processes for breaking down hemicellulose, which is a major component of the cell wall of plants, are also disclosed, including methods and processes for hydrolyzing hemicelluloses in any plant or wood or wood product, wood waste, paper pulp, paper product or paper waste or byproduct. In addition, methods of designing new xylanases, mannanases and/or glucanases and methods of use thereof are also disclosed. The xylanases, mannanases and/or glucanases have increased activity and stability at increased pH and temperature.
U.S. Patent Application Publication No. 2009/0220480 discloses polypeptides having any cellulolytic activity, e.g., a cellulase activity, an endoglucanase, a cellobiohydrolase, a beta-glucosidase, a xylanase, a mannanse, a β-xylosidase, an arabinofuranosidase, and/or an oligomerase activity, polynucleotides encoding these polypeptides, and methods of making and using these polynucleotides and polypeptides. The disclosed polypeptides can be used in a variety of pharmaceutical, agricultural, food and feed processing and industrial contexts. Compositions or products of manufacture comprising mixtures of enzymes comprising at least one enzyme of the invention are also disclosed.
There still remains a need for improved degradation of biomass, especially recalcitrant cellulosic biomass. For example, it would be highly beneficial to have bio-engineered, designer cellulosomes showing highly synergistic, improved degradative capabilities.